PROJECT SUMMARY Glioblastoma is among the most lethal of all cancers and the most common primary malignant brain tumor, typically lethal by 15 months. After initial tumor resection, we rely on an imperfect measure of disease response, namely magnetic resonance brain imaging, as obtaining tumor tissue at recurrence is often dangerous or fails to impact survival and is rarely performed. Indeed, many patients rely only on a tiny biopsy for initial diagnosis with no ability for subsequent deep molecular analyses, let alone monitoring of disease over time. The current proposal will address a fundamental gap in the field of GBM, i.e. the lack of predictors not dependent on the availability of tumor tissue and those that can provide real-time, tissue-free tumor monitoring, which is particularly critical in monitoring treatment response and assessing disease progression longitudinally. Blood-based biomarkers such as circulating tumor cells and cell free DNA are attractive, but have yet to provide meaningful results in GBM (compared to other cancers) and have yet to become clinical diagnostics. Tumor educated platelets (TEPs) has recently been reported and shown to contain tumor-specific gene expression signatures that are associated with outcome for several tumor types, but the clinical value and the mechanisms of enrichment of TEPs are unknown. The overall goals of this proposal are to elucidate the mechanism underlying the tumor cell?platelet interaction that leads to TEPs and to develop a TEP-based liquid biopsy platform for longitudinal monitoring of disease in patients with GBM. In this project, we will test the hypothesis that platelet numbers are increased in response to tumor signaling in the bone marrow and that platelets take up tumor- derived extracellular vesicles containing RNA cargo in a PDPN dependent manner. TEP RNA signatures reflect underlying tumor biology, growth, and treatment resistance, and will be utilized as biomarkers. In Aim 1, we will test the hypothesis that pro-inflammatory cytokine-mediated paraneoplastic thrombocytosis promotes formation of TEP signatures in GBM. In Aim 2, we will test the hypothesis that PDPN interaction with the CLEC2 receptor contributes to transfer of RNA from tumor cells to platelets. In Aim 3, we will test the hypothesis that TEP RNA signatures can be used to monitor disease burden longitudinally in patients with GBM.